Data from: "Single capsid mutations modulating phage adsorption, persistence, and plaque morphology shape evolutionary trajectories in ΦX174"

Zip File "Data_sets_Phage_titers" Raw phage and bacteria countings (i.e. plaque-forming units [PFU] for phages, colony-forming units [CFU] for bacteria) and plaque morphology data for data analysis and visualisation in the code found in https://github.com/OReyesMatte/Reuter_2025/tree/main/Data_analysis. The folder contains nine csv files of a total of seven experiments, which are described in detail below. Information about the data columns of each file is mentioned below, as well as in the code file. Figure numbers for which the respective data were used are indicated for each experiment. Phage genotypes indicated in the data sets refer to the codon number. In the final manuscript, the amino acid number was used instead, shifting all codon numbers by -1 due to the removal of the first methionine in the proteins. In the following, the individual experiment files are described in detail: Experiment 1: 1. File “experiment _1_ 3_h_transfers.csv” - In the experiment, five independent selection lines, all initiated from ancestral phage PhiX174, we evolved using serial transfers with a 3-h transfer time until a heritable small-plaque phenotype was observed. Transfers we stopped as soon as the first heritable small plaques could be isolated. - Counting data of bacteria and phages were recorded for each transfer and selection line to monitor and adjust the MOI input. Specification of each column in the file “experiment _1_ 3_h_transfers.csv” - Transfer (T): Individual three-hour transfers until the first heritable small plaques could be isolated - Line (L): five selection lines started at the beginning of the experiment from five individual large plaques of ancestral, large-plaque-forming phage PhiX174 - CFU: colony-forming units of E. coli C after 2 hours of growth to the exponential phase. CFU count of T3 (644 CFU) was acquired by counting a quarter of the plate (161 CFU), which was multiplied by a factor of 4 - Dilution_CFU: plating dilution used to plate the colonies counted; 100 µl of this dilution was plated unless stated otherwise - PFU: Plaque-forming units counted on plaque assay plate. Plaque assays were done for the phage lysate extracted after each transfer by chloroform extraction - Dilution_PFU: plating dilution used to plate phage plaques. 100 µl of this dilution was plated unless stated otherwise 2. File “experiment_1_3_h_transfers_small_plaque_frequency.csv” - In this file, the number of verified small-plaque-forming phage particles compared to the total number of phages is recorded for the first transfer, at which heritable small plaques could be detected and isolated - This data was used to estimate the frequency of small plaque mutants in the population —> The data set was used to generate supplementary figure Fig. S4A Specification of each column in the file “experiment_1_3_h_transfers_small_plaque_frequency.csv” - Line (L): Five selection lines started at the beginning of the experiment from five individual large plaques of ancestral PhiX174 - Genotype: Genotype of the small plaque mutant isolated from the 3-h regime, with the single-point mutation indicated as F [for F protein] and in the brackets the mutation in the protein with old amino acid, amino acid position, and new amino acid - Transfer_number (T): Number of 3-h transfers required to isolate one isogenic, heritable small plaque - Total_PFU_ml: Total Phage concentration in PFU per ml for the transfer at which the first small plaques were observed. Column corresponds to the PFU_ml column of data set "experiment_1_3_h_transfers.csv"* (see above) for the row with the transfer number at which the first heritable small plaques could be isolated: - For Line L1, the first small plaques were isolated after transfer T6 - For Line L2, the first small plaques were isolated after transfer T7 - For Line L3, the first small plaques were isolated after transfer T5 - For Line L4, the first small plaques were isolated after transfer T4 - For Line L5, the first small plaques were isolated after transfer T6 - Small_PFU_ml: Estimated concentration of small plaques of the "Total_PFU_ml” Experiment 2: 3. File “experiment_2_30_min_transfers.csv” - In the experiment, five independent selection lines, all initiated from ancestral phage PhiX174, we evolved using serial transfers with a 30-minute transfer time for fifty transfers. - Each passage day consisted of four 30-minute transfers (T1-T4) followed by one 3-hour amplification step (T5). The experiment was done for a total of ten passage days. - After transfers T1-T4 of each day, phages were quantified using a spot test. After each amplification step (T5 on each day), phages were titered using a plaque assay after chloroform extraction. Phage titers were used to monitor and adjust the MOI input. Specification of each column in the file “experiment_2_30_min_transfers.csv” - Passage (P): every passage day consisted of four consecutive 30-min transfers (T1 to T4) followed by one 3-hour-long amplification step (T5) after which phages were extracted by chloroform-extraction. P2 had to be repeated due to a failure to produce detectable phage particles. Passage P2b was worked and was used as the second passage day. In total, 10 passage days (40 30-minute transfers + 10 amplification steps) were done - Transfer (T): Individual transfers + amplification step done within one passage day - Passage_ID: Individual ID for each single passage+transfer ranging from 1-50 - Line (L): 5 selection lines started at the beginning of the experiment from five individual large plaques of ancestral, large-plaque-forming phage PhiX174 - CFU: colony-forming units after 2 hours of E. Coli C growth to get an exponential overday culture - Dilution_CFU: plating dilution used to plate the colonies counted; 100 µl of this dilution was plated unless stated otherwise - CFU_ml: colony-forming units per millilitre calculated by CFU* Dilution_CFU*10. Factor 10 for multiplication was used because the plating volume was 100 µl - PFU: Plaque-forming units counted on the plaque assay plate (only done for transfer 5 of each passage) - Dilution_PFU: plating dilution used to plate phage plaques. 100 µl of this dilution was plated unless stated otherwise - PFU_ml: Plaque-forming units per millilitre calculated by PFU* Dilution_PFU*10. Factor 10 for multiplication was used because the plating volume was 100 µl - Spot_assay_dilution: 10-fold dilution of phages from transfers T1 to T4, at which a spot on a lawn of ancestral, phage-sensitive E. Coli C could be detected. (Only done for transfers 1 to 4 for each passage) - Output_phage_ml: Phage concentration in phages per millilitre calculated after each transfer. For transfers T1 to T4, by calculating Spot_assay_dilution*500. Factor 500 because 2 µl of phage lysate was plated. For transfer T5, concentration calculation as described under column “PFU_ml”. -Inoculation_volume_ml: Volume of phage lysate put into each transfer. For transfers T2 to T5 on each passage day, 100 µl of phage+bacteria mix from the previous transfer was used. For the first transfer of each passage day, the phage input volume was calculated to get an MOI input of ~0.1. - Real_virus_concentration: Real phage concentration put into each transfer based on Spot assay and PFU counts - Real_MOI: Real MOI input calculation for each transfer based on real virus concentration from column "Real_virus_concentration" and bacteria concentration in relation to colony-forming units per ml determined by plating bacteria colonies (recorded in column "CFU_ml"). Experiment 3: 4. File “experiment_3_adsorption_assay.csv” - This adsorption assay was used to determine the adsorption dynamics of four different phage genotypes (ancestor, large-plaque mutant F(T101A), small-plaque mutant F(G322D), and small-plaque mutant F(S427*)) - Based on the quantified phage titers during a 6-minute adsorption period, adsorption curve slopes were estimated, which were then used to calculate adsorption rate constants. —> The data set was used to generate figure Fig. 3A and Supplementary Figure S.9 Specification of each column in the file “experiment_3_adsorption_assay.csv” - Genotype: Phage genotypes for which the adsorption assay was done: - wild type (WT) - small-plaque mutant G322D evolved in Experiment 1: 3-hour transfers - small-plaque mutant S427* evolved in Experiment 1: 3-hour transfers - large-plaque mutant T101A evolved in Experiment 2: 30-min transfers - Run: Repeated experimental runs, different runs are biological replicates, run on independent bacterial cultures started from independent colonies. Different number of runs for each phage genotype - Wild type: Runs 1,2,3,4,5,6,7 - T101A: Runs 5,6,7, - G322D: Runs 1,2,3,4,5,6,7 - S427*: Runs 1,2,3,4,5,7 - Sampling_time_min: Sampling time point in minutes during the adsorption period from time point 2 minutes to 6 minutes in 2-minute time intervals. - PFU_1: Plaque-forming unit counts (PFU) for the first plating of triplicate plating for phage lysate for each sampling time point - PFU_2: Plaque-forming unit counts (PFU) for the second plating of triplicate plating for phage lysate for each sampling time point - PFU_3: Plaque-forming unit counts (PFU) for third plating of triplicate plating for phage lysate for each sampling time point - Median_PFU: Median PFU counts from triplicate plating PFU_1, PFU_2, PFU_3 - Predilution: After sampling at the adsorption time point 2 min to 6 min, 500 µl of sample was diluted in 4.5 ml of unsupplemented LB-medium cooled on ice to prevent infection cycle completion and secondary adsorptions, resulting in a 10-fold dilution of the sample. - Dilution_plating: Plating dilution used to plate 100 µl of pre-diluted phage lysate from each sampling time point. - Dilution_in_ml: Dilution step to calculate phage concentration in PFU/ml. Factor is 10x because 100 µl of phage lysate was used for plating. - PFU_ml: Phage concentration in plaque-forming units per ml (PFU/ml), calculated as Median_PFU * Predilution * Dilution_plating * Dilution_in_ml - CFU: Colony-forming units of wild-type E. coli C at the beginning of the adsorption period - Dilution_CFU: Plating dilution used to plate CFUs; 100 µl of this dilution was plated for colony counts unless stated otherwise. - CFU_ml: Bacteria concentration in colony-forming units per ml CFU/ml) at the start of the adsorption period. Calculated as counting colony-forming units (CFU) * Dilution_CFU * 10 (because 100 µl of Dilution_CFU were used for plating) Experiment 4: 5. File “experiment_4_plaque_image_analysis.csv” - This file contains the plaque morphology data extracted after segmentation. - Plaque area and turbidity information were extracted for individual plaques of four independent plaque assay plate replicates for four different phage genotypes (ancestor, large-plaque mutant F(T101A), small-plaque mutant F(G322D), and small-plaque mutant F(S427*)) - Information about the segmentation is found under https://github.com/OReyesMatte/Reuter_2025/tree/main/Plaque_analysis. —> The data set was used to generate figure Fig. 3B and supplementary figure Fig. S5 Specification of each column in the file “experiment_4_plaque_image_analysis.csv” - Block: Experimental block during which the phage isolate was evolved - 0: wild type non-evolved - A: 30-minute transfer experiment - 1: 3-hour transfer experiment - Phenotype: Lysis plaque phenotype forming large plaques (LP) or small plaques (SP) - Line: Selection line phage was evolved in - WT: wild type non-evolved - L3: 30-minute transfer experiment (corresponding to genotype F(T101A)) - L1: 3-hour transfer experiment (corresponding to genotypes F(G322D) [L1]) L4: 3-hour transfer experiment (corresponding to F(S427*) [L4]) - Image: Replicate image of individual plaque assay plate, for each phage isolate, four images labelled A to D were taken (corresponding to four independent biological replicates done on different indicator bacteria cultures) - Colony: Individual ID of a lysis plaque on a plaque assay plate selected from the segmentation. Plaques were manually selected, checking for correct recognition of the mask (i.e. intact area, non-connected, individual plaques) - Area: Lysis plaque area in cm^2 - Distance: Distance to the plaque periphery in pixels (px) - Intensity: Plaque intensity in pixels (px) at individual distance position within the plaque - Intensity_SD: Standard deviation of pixel intensity - ID: Unique identifier combining block, phenotype, and line Experiment 5: 6. File “experiment_5_30min_free_phages.csv” - This file contains counts of bacterial colonies and free phage particles for multiple 30-minute transfers for phage genotypes ancestor (WT) and mutant F(T101A) - Free phage particles were quantified after each 30-minute transfer by filtration and plaque assay plating. —> The data set was used to generate figure Fig. 5C Phage titers of free phages after every 30-minute transfer were quantified separately for the ancestor and large-plaque mutant in Experiment 5 using sterile filtration and plaque assay titering in multiple biological replicates to improve the accuracy of the titer quantification after each 30-min transfer. Specification of each column in the file “experiment_5_30min_free_phages.csv” - Replicate: Biological replicates of the assay. In total, 3 biological replicates were done. For each replicate, a different bacterial culture initiated from an individual colony was used. -Treatment: Treatment Filtration: Samples were sterile filtered using a 0.2 µm filter to remove bacteria and phages still inside bacterial hosts -Transfer: Transfer identity: Transfers T1, T2, T3, and T4 were individual 30-minute transfers. For transfer T1, phage lysate was used to inoculate bacterial hosts at an MOI input of ~0.1. For the three consecutive transfers, 100 µl of phage-bacteria mix was transferred onto fresh, susceptible ancestral bacteria cells. The same protocol was used in Experiment 2 (30-min transfer regime). -Line: Genotype used to initiate the transfer - WT: ancestral wild-type phage genotype, large plaque former - T101A: mutant T101A forming large plaques, evolved during 30-minute transfers in Experiment 2: 30-min transfer regime. -CFU: Colony-forming units of the ancestral E. coli C culture in exponential phase at the moment of infection initiation -Dilution_CFU: Plating dilution used to plate CFUs; 100 µl of this dilution was plated for colony counts unless stated otherwise. -CFU_ml: Colony-forming units in ml, calculated by CFU*CFU_dilution*10 (because 100 µl were plated) -PFU_LP_1: Plaque-forming units of the large-plaque phenotype for each sample of replicate plating 1 -Dilution_1: Plating dilution used for PFU counts, 100 µl of plating dilution was plated for plaque counting of replicate plating 1 -PFU_LP_2: Plaque-forming units of the large-plaque phenotype for each sample of replicate plating 2 -Dilution_2: Plating dilution used for PFU counts, 100 µl of plating dilution was plated for plaque counting of replicate plating 2 -PFU_LP_3: Plaque-forming units of the large-plaque phenotype for each sample of replicate plating 3 -Dilution_3: Plating dilution used for PFU counts, 100 µl of plating dilution was plated for plaque counting of replicate plating 3 -PFU_ml_LP1: Plaque-forming units per ml of large plaque phenotype calculated as PFU*dilution_1*10 (because 100 µl were plated) of replicate plating 1 -PFU_ml_LP2: Plaque-forming units per ml of large plaque phenotype calculated as PFU*dilution_1*10 (because 100 µl were plated) of replicate plating 2 -PFU_ml_LP3: Plaque-forming units per ml of large plaque phenotype calculated as PFU*dilution_1*10 (because 100 µl were plated) of replicate plating 3 -Median_PFU_ml: Median large plaque PFU per ml calculated based on phage concentrations of the three replicate platings -Median_WT: Median of titering for the wild type taken from column "Median_PFU_ml" used to calculate the ratio between free phages of mutant F(T101A) and the ancestor The data was also used to calculate and visualise input phage concentrations into each 30-minute transfer for the large-plaque-forming mutant F(T101A) and the ancestor. After review, supplementary figure Fig. S7 was included to visualise titer differences between the beginning and end of a 30-min transfer. Experiment 6: 7. File “experiment_6_decay_analysis.csv”’ - This file contains counting data of bacteria colonies and phage plaques during a long (5 hours) host-limited infection period for four phage genotypes (ancestor, large-plaque mutant F(T101A), small-plaque mutant F(G322D), and small-plaque mutant F(S427*)) - This data was acquired to analyse phage population dynamics during long infection periods, with particular focus on the decay period. —> The data set was used to generate figure Fig. 4B and supplementary figure Fig. S6 Specification of each column in the file “experiment_6_decay_analysis.csv” - Sampling_time_min: Sampling time in minutes, phages were extracted by chloroform extraction - Genotype: Genotypes of phage isolates - WT: wild-type large plaque former - T101A: large plaque mutant evolved from WT using 30-min transfers - G322D: small plaque mutant evolved from WT using 3-hour transfers - S427*: small plaque mutant evolved from WT using 3-hour transfers - CFU: colony-forming units of E. coli C exponential phase culture at the moment of phage infection - Dilution_CFU: Plating dilution used to plate CFUs; 100 µl of this dilution was plated for colony counts unless stated otherwise. - CFU_ml: Colony-forming units per ml calculated by CFU*CFU_dilution*10; factor *10 because 100 µl of CFU_dilution was plated - PFU: Plaque-forming units - Dilution_PFU: Plating dilution used for PFU counts, 100 µl of plating dilution was plated for plaque counting - PFU_ml: Plaque-forming units per ml calculated by PFU*PFU_dilution*10; factor *10 because 100 µl of PFU_dilution was plated Experiment 7: 8. File “experiment_7_OSGC_WT.csv” - This file contains counts of bacterial colonies and phage plaques during a one-step growth curve experiment on ancestral phage PhiX174 - Phage titers were quantified for one infection cycle, including the eclipse and rise period, to estimate burst size and lysis time. —> The data set was used to generate supplementary figure Fig. S3 Specification of each column in the file “experiment_7_OSGC_WT.csv” - Genotype: Phage genotype for which OSGC was done: ancestral phage PhiX174 (WT) - MOI: Multiplicity of infectiousness input when adsorption is initiated - Sampling_time_min: Sampling time point in minutes after the 6-minute synchronised adsorption period, followed by a 2000x dilution step - PFU_1: Plaque-forming unit counts (PFU) for the first plating of triplicate plating for phage lysate for each sampling time point - PFU_2: Plaque-forming unit counts (PFU) for the second plating of triplicate plating for phage lysate for each sampling time point - PFU_3: Plaque-forming unit counts (PFU) for the third plating of triplicate plating for phage lysate for each sampling time point - Median_PFU: Median PFU counts from triplicate plating PFU_1, PFU_2, PFU_3. If triplicate plating was not possible, the mean of two platings or just one plating value was taken for phage titer calculation. The reason for this was not enough sampling volume to do triplicate plating (i.e. at early time points). - Dilution_PFU: Plating dilution used to plate phage lysate isolated after the one-step growth curve using chloroform in a plaque assay - Dilution_OSGC: Dilution step between adsorption and OSGC sampling to prevent secondary infection. The dilution step for all three one-step runs was a 2000-fold dilution using 10 µl of bacteria-phage mix after adsorption and diluting it into 20 ml supplemented, prewarmed (37 °C) LB medium. - PFU_ml: Phage concentration in plaque-forming units per ml (PFU/ml), calculated from Median_PFU *Dilution_PFU - t0_PFU_ml: Phage concentration in PFU/ml at time point t0 after adsorption. These are free phages which need to be subtracted from each following time point phage titer to normalise the data for free/unadsorbed phages. - PFU_t0_norm: Normalised PFU concentration for each time point calculated by PFU_ml - t0_PFU_ml - Bacteria_CFU_ml: Bacteria concentration in colony-forming units per ml CFU/ml) at the start of the adsorption period. Calculated by counting colony-forming units (CFU)* Plating dilution (10^6) - Infected_CFU_ml: Number of Bacteria which could be, in theory, infected by phages, calculated by Bacteria_CFU_ml*MOI/Dilution_OSGC - PFU_ml_produced: Mean phage burst size at each sampling time point - Elapsed_time: Elapsed time since adsorption. Calculated by adding 6 min adsorption to each "Sampling_time_min" OSGC sampling time point 9. File “experiment_8_phage_titer_WT_30_180.csv” This file contains counts of bacterial colonies and phage plaques at 0 minutes, 30 minutes, and 180 minutes post-infection of an ancestral phage PhiX174 The file was included after the manuscript revision process to add an additional supplementary figure showing the variability of phage titers between different replicates within a sampling time point. Data of Replicate 1 is found in file "exp_6_decay_analysis.csv" as it was also used to visualise phage population dynamics and decay in figures Fig. 5C and Fig. S6. -—> The data set was used to address a reviewer comment but was not included as an additional figure in the final manuscript Specification of each column in the file “experiment_8_phage_titer_WT_30_180.csv.” - Replicate: Biological Replicate done on individual bacteria cultures of ancestral, phage-sensitive E.coli C. Replicate 1 data was used from file "Experiment 6: Decay analysis" - Sampling_time_min: Sampling time in minutes, phages were extracted by chloroform extraction, either 0 minutes, 30 minutes or 180 min post infection initiation - Genotype: Genotypes of phage isolates - WT: wild-type large plaque former - CFU: colony-forming units of E. coli C exponential phase culture at the moment of phage infection - Dilution_CFU: Plating dilution used to plate CFUs; 100 µl of this dilution was plated for colony counts unless stated otherwise. - CFU_ml: Colony-forming units per ml calculated by CFU*CFU_dilution*10; factor *10 because 100 µl of CFU_dilution was plated - PFU: Plaque-forming units - Dilution_PFU: Plating dilution used for PFU counts, 100 µl of plating dilution was plated for plaque counting - PFU_ml: Plaque-forming units per ml calculated by PFU*PFU_dilution*10; factor *10 because 100 µl of PFU_dilution was plated. For time point t= 0 minutes, phages were added at ~10^7 PFU/ml from a stock. Hence, the phage concentration is stated the same for all three replicates.